Background- In the vertebrate retina, six types of neurons and Muller glia originate from pools of retinal progenitor cells. Extracellular signals and intrinsic regulatory mechanisms dictate cell-type specification. We are using both system-based and reductionist approaches to investigate complex molecular pathways controlling development and function of retinal neurons, especially photoreceptors (6). To generate photoreceptor gene regulatory networks (GRNs), we first performed genome-wide expression analysis (by microarray and RNA-seq) of flow-sorted rods isolated from developing and mature mouse retina. The transcriptome data was also obtained by perturbing a key regulatory node, i.e., Neural Retina Leucine zipper (Nrl) in Nrl-/- photoreceptors. We have previously generated NRL-targetome data by ChIP-seq and are now producing finer maps by ChIP-nexus technology. We have also generated genome-wide profiles of multiple histone modifications (e.g, H3K4me3 and H3K27me3), DNA methylation and chromatin accessibility (ATAC-seq). Our bioinformatics team is assembling and integrating all NGS data sets to produce control networks associated with rod photoreceptor differentiation, maturation and aging. We have built a template database (Ret-seq) that will include gene expression and epigenetic patterns from the retina and photoreceptors of different organisms, tissues, and experimental conditions to facilitate data sharing and for comparative system-level approaches. We have initiated a large program of transcriptome and epigenome profiling of disease (human, mouse and stem cell-based) -see EY000546 and EY000474. Specification of photoreceptor cell identity- Our main research focus is the NRL-centered GRN that determines rod photoreceptor development and maturation. NRL controls photoreceptor fates by inducing rod development and suppressing cone differentiation. However, what controls NRL and balances rod and cone fates remains unclear. We have previously shown that Otx2 plays an essential role in maintaining Nrl expression in developing rods to promote rod fate and maturation (4) and that the retinoid-related orphan receptor beta gene (Rorb) is required for Nrl expression (Liu H et al. 2013). However, Rorb differentially expresses two isoforms: RORbeta2 in photoreceptors and RORbeta1 in photoreceptors, progenitor cells, and other cell types. By deleting RORbeta2 or RORbeta1, the cone:rod ratio increases, whereas when both isoforms are deleted in Rorb-/- mice, cone-like cells are produced almost exclusively at the expense of rods, suggesting that both isoforms induce Nrl. Rescue experiments using either RORbeta; isoform in Rorb-/-, but not in Nrl-/- retina explants, reactivated Nrl and rod genes. Thus, Nrl is the effector for both RORbeta isoforms in rod differentiation. Loss of RORbeta2 expression in Nrl-/- mice and activation of the RORbeta2-specific promoter by NRL suggest the existence of a feedback activation loop between Nrl and Rorb genes that may reinforce fate commitment to rod differentiation (1). In mice, developing cones undergo a round of specification that generates short-wave-sensitive (S) and medium-wave-sensitive (M) cones. The general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1) can function as either an activator or repressor depending on the target gene and context. In photoreceptors, GTF2IRD1 binds to enhancer and promoter regions in the mouse rhodopsin, M- and S-opsin genes. In association with CRX and NRL, it enhances rhodopsin expression. Through interaction with CRX and thyroid hormone receptor beta 2 (Trb2), it enhances M-opsin expression whereas it suppresses S-opsin expression. Null mutation leads to aberrant S-opsin overexpression and M-opsin underexpression in M cones, suggesting a role in regulating the level and topology of rod and cone gene expression and maintaining M cone cell identity and function as well as rod function (3). While examining photoreceptor transcriptome and epigenome data from wild type (rods) and Nrl-/- (S-cone like) mouse retina, we discovered genetic and epigenetic vestiges of short wavelength (S), but not medium wavelength (M), cones in developing rods. Multiple lines of evidence, including lineage tracing using different mouse Cre-lines, provided strong support in favor of the genesis of rods from cells expressing S-cone opsin. However, such a molecular footprint of S-cones was not observed in zebrafish rods. We then investigated the origin of rods in extant vertebrates within evolutionary-developmental (evo-devo) paradigm. Our data invoke novel questions regarding the evolution of generally rod-dominant duplex retina in mammals and support the interpretation wherein the S-cone lineage was recruited, via NRL, to augment rod photoreceptors and overcome the nocturnal bottleneck in early evolution (Kim et al. submitted). Establishment of photoreceptor morphology- Maturation of rod photoreceptors requires acquisition of unique morphological features, e.g., cell polarity, outer segments and synapses. In the current FY, we have completed several studies related to polarity and cilia biogenesis, and established the following: 1) Prickle1 is a key component of the Wnt-signaling pathway that regulates establishment of planar cell polarity and is associated with establishment of apical-basal polarity as well (Liu et al. 2014, in collaboration with Dr Tiansen Li). This project is now being pursued by Dr. Chunqiao Liu in his lab in China; 2) A rod-specific target of NRL, Reep6.1 (2), plays a role in trafficking and docking of vesicles carrying cargoes to the outer segment and synaptic regions of rod photoreceptors; 3) Cc2d2a is essential for the assembly of sub distal appendages, which anchor cytoplasmic microtubules and prime the mother centriole to initiate cilia formation, a pre-requisite for outer segment biogenesis (5); 4) The centriolar coiled coil protein CP110 is implicated in cilia vesicle docking, an essential early step in cilia formation. CP110 also acts as a suppressor to control the timing, while at the same time promoting, ciliogenesis. Since Dr. Tiansen Li in NNRL is pursuing polarity and cilia projects, we have decided to focus on photoreceptor synapses. Rod and cone photoreceptors have ribbon synapses with distinct presynaptic morphology; rods have spherules with a single ribbon, whereas cones have pedicles with multiple ribbons. Based on the observation that synaptic terminals in Nrl-/- retina switch from spherule to pedicle-like morphology, we hypothesized that downstream targets of NRL are determinants of spherule morphogenesis. Of 200 candidate genes based on transcriptome data, 75 selected NRL targets are being evaluated by analyzing the retinal phenotype after in vivo electroporation of corresponding shRNA. We have already identified several genes that alter rod spherule morphology when knocked down in early development. Aging retina, mitochondria and disease- Expression profiling of aging rod photoreceptors has revealed significant changes occurring in mitochondria, suggestive of functional deficits that accumulate over time. To investigate the role of mitochondria in photoreceptor aging, we first developed a method by adapting an existing in vitro assay. Mitochondrial function is determined as oxygen consumption rate in freshly isolated retina punches using the Seahorse, HF24 Analyzer. Our data suggest that mitochondria in young, healthy retina function at maximum capacity (with little reserve capacity) and would therefore be most sensitive to even small changes such as those occurring in aging or in pre-disease conditions.